The immune responses to Pneumocystis are poorly understood, but cytokines may play a role in both clearing Pneumocystis infection and in the hypoxia associated with Pneumocystis pneumonia that may be exacerbated following initiation of therapy. We are using the scid mouse model, as well as other immunodeficient mice, to further evaluate the role of individual cytokines and other immunoregulatory molecules in modulating Pneumocystis infection. We have developed a real time PCR assay to quantitate Pneumocystis over a wide dynamic range, and are currently examining Pneumocystis infection in healthy animals to better understand immune responses in the normal host. Using microarrays, we have been able to identify genes that are differentially expressed during Pneumocystis infection in healthy compared to immunodeficient mice; the majority of differentially expressed genes are related to the immune system. Immunohistochemical studies of the lungs done in parallel have demonstrated that there is an influx of CD4 cells and macrophages at 5 weeks, and, surprisingly, an influx of B cells at 6 weeks in immunocompetent mice but not CD40L KO mice. Microarray studies suggest that the B cells persist through at least 10 weeks, well after the infection has been cleared. We have extended these studies to examine immune responses in other immunodeficient mice, including those with defective innate immune responses, such as Myd88 knockout mice which are deficient in TLR signals, and CDld KO mice, which lack NKT cells. Our studies have shown that these mice are not susceptible to uncontrolled Pneumocystis infection, suggesting that neither TLR signaling, nor NKT cells are critical to controlling Pneumocystis infection. In collaboration with Philip Murphy, Michail Lionakis, and their groups, we have examined changes in expression of a variety of chemokine and chemokine receptor genes, using Taqman real-time PCR assays, and have been able to identify CCR2 and its ligands as potentially important in the response to Pneumocystis infection. However, in examining the susceptibility of CCR2 knock-out mice to Pneumocystis infection, we found that they are able to clear infection similar to wild-type animals. We also examined CX3CR1 knock-out mice for susceptibility to Pneumocystis infection, since CX3CR1 and its ligand identify alternately activated macrophages and are critical to clearance of another fungus, Candida, in mouse models. These mice were again able to clear Pneumocystis infection with kinetics similar to wild-type mice. Using microarray analysis of purified CD4 cells isolated from lungs of Pneumocystis-infected mice, we were able to show that CXCR6 is preferentially upregulated compared to cells from uninfected animals, and by Q-PCR that both CXCR6 and its ligand, CXCL16, are unregulated in lung tissue from immunocompetent Pneumocystis-infected mice. To allow us to evaluate immune responses more easily, we have developed a multiplex assay that allows the evaluation of expression levels of 30 genes simultaneously, using branched DNA amplification combined with Luminex bead technology. We are using this assay to examine the lungs of various strains of knock-out mice at different stages of Pneumocystis infection, to determine if their immune responses are similar to those seen in healthy, immunocompetent mice. We are examining the kinetics of immune responses in the lung of healthy animals exposed to Pneumocystis, using flow cytometry. We have found that gamma interferon, and less predictably, IL-17, expression are increased in lymphocytes of the lung, primarily CD4 cells, during Pneumocystis infection and clearance. In follow-up, we found that IL-17A knockout mice are able to clear Pneumocystis infection, thus demonstrating that IL-17 is not critical to control of Pneumocystis infection. We are also examining the response of dendritic cells to the most abundant Pneumocystis antigens, the major surface glycoprotein (MSG). Finally, we are performing reconstitution studies in CD40KO mice, to try to identify which cells are critical to the mounting an effective immune response against Pneumocystis. For these studies we use CD45 allelic variants as donors to allow us to track transferred cells. We have been able to show that unfractionated spleen cells from immunocompetent mice are able to clear Pneumocystis infection, and are currently examining which subpopulations of cells can confer this protection. Preliminary studies suggest that B cells play an important role in conferring long-lasting protection. We are further examining the role of B cells by performing immune reconstitution studies using B cells that are quasimonoclonal, to help determine if antibody production or, alternatively, other properties of B cells, such as antigen presentation, are critical to controlling Pneumocystis infection. It is hoped that these studies will provide insights into the mechanisms critical for control of Pneumocystis infection in the healthy host, and may provide mechanisms for increasing clearance of Pneumocystis or decreasing the inflammation that may be causing hypoxia.